Antimicrobials are widely used to maintain animal health and productivity; however, their misuse contributes to antimicrobial resistance (AMR), a critical One Health and global health concern. This study assessed the knowledge, awareness, and practices of animal health practitioners regarding antimicrobial use and AMR in Niger State, Nigeria. A questionnaire-based cross-sectional survey was conducted using purposive sampling, and data were analyzed at a 95% confidence level. A total of 62.8% of targeted practitioners participated. About 42.3% used antimicrobials for treating infections, while 12.2% used them for growth promotion. Only 19.5% correctly identified misuse as under-dosing, and 13.0% were unaware of what misuse entailed. Although 69.1% adhered to daily dosages as prescribed, only 1.6% performed confirmatory diagnoses before administration. Tetracycline (99.2%) and penicillin-streptomycin (82.1%) were the most frequently used antimicrobials. Satisfactory knowledge of antimicrobial use was significantly associated with practitioners aged 50–59 years (p = 0.002) and veterinarians (p = 0.001). Factors influencing misuse included improper usage (p < 0.001), weak regulation (p = 0.005), poor client finances (p = 0.001), low expertise (p = 0.001), and counterfeit drugs (p = 0.001). The findings underscore the urgent need for antimicrobial stewardship, stricter regulation, and continuous education of animal health practitioners.